One of the most elusive goals in hematologic research has been to understand how hematopoietic stem cells (HSC) self-renew, since this knowledge could be applied in protocols to expand clinically useful numbers of HSC for bone marrow transplantation and targeted gene therapy for hematologic disorders. A number of studies have suggested an essential role for homeodomain-containing proteins belonging to the Hox gene family in the regulation of adult HSC self-renewal. Experiments where the homeobox gene, Hoxb4, was overexpressed using a retroviral vector in HSC resulted in a 40-fold expansion in long-term repopulating HSC (LT-HSC) numbers in vitro. A similar stimulation of LT-HSC symmetric self-renewal in vitro was observed when Hoxb6 or Hoxa9 were overexpressed in LT-HSC. Loss of Mll, a mammalian trithorax homolog that functions to maintain Hox gene expression, results in the loss of adult LT-HSC in vivo, which further supports the hypothesis that Hox gene activity is essential for HSC self-renewal. Recent experiments where the acute myeloid leukemia oncoprotein NUP98-HOXA10 was expressed in murine hematopoietic progenitor cells using a retroviral vector showed an unprecedented 10,000-fold expansion of LT-HSC over a two-week in vitro culture period. In studies outlined in this proposal, we have observed a similar 10,000-fold in vitro expansion of adult LT-HSC using a related Hox fusion protein, NUP98-HOXA9. The major objective of this proposal will be to delineate the molecular mechanisms responsible for the dramatic enhancement of LT-HSC symmetric self-renewal mediated by NUP98- HOXA9 using genetic and biochemical approaches. In Aim 1, we will functionally test whether genes commonly up-regulated in LT-HSC by NUP98-HOXA9, Hoxb4, and Hoxa9 contribute to NUP98-HOXA9- mediated symmetric self-renewal. For Aim 2, we will characterize the extent to which Hoxa5 and Hoxb5 contribute to the potent HSC self-renewal phenotype stimulated by NUP98-HOXA9. Hoxa5 and Hoxb5 are the two most highly up-regulated Hox genes in LT-HSC expressing NUP98-HOXA9 so these paralogous genes may have a unique function in stimulation of LT-HSC expansion. Finally, in Aim 3 we will define the roles of the Hox co-factors, Pbx1 and Pbx3, in LT-HSC self-renewal mediated by Hoxb4 and NUP98- HOXA9 and identify NUP98-HOXA9-interacting proteins in LT-HSC using mass spectrometry. Our ability to expand LT-HSC 10,000-fold in vitro using NUP98-HOXA9 will allow, for the first time, conventional biochemistry to be done using a pure population of LT-HSC. This will facilitate the biochemical characterization of Hox protein complexes that are functioning in LT-HSC to regulate the self-renewal process. Collectively, these studies will provide new insights into the genes and pathways controlling adult LT-HSC symmetric self-renewal that is stimulated by Hox gene activity.